Neovascular diseases of the eye include retinopathy of prematurity, proliferative diabetic retinopathy, and the exudative or wet form of age-related macular degeneration (AMD). Together these diseases affect all age groups and are the leading causes of vision impairment in developed nations. The collective evidence suggests that the vascular endothelial growth factor (VEGF) family is critical for ocular angiogensis and this has become a major target for therapeutic intervention. Investigation of VEGF action has largely focused on receptor binding events at or near the plasma membrane and subsequent activation of classical signal transduction cascades. However, it is now apparent from our work and others that the signaling mediated by ligands binding to the VEGFRs (VEGFR1 and VEGFR2) is much more complex and involves intracellular trafficking of VEGFRs. Our data shows that targeted subcellular translocation of VEGFRs to adherens/tight junctions (AJs/TJs) results in the VEGFRs regulating vascular permeability or, if translocated to the nucleus VEGFRs can regulate transcription of pro- and anti-angiogenic regulators. Preliminary data also indicate that the specific ratio of VEGFR1:VEGFR2 at specific cell sites (such as AJs/TJs and the nucleus) is critical in determining vascular permeability and angiogenesis. Furthermore, endosomal sorting, -secretase and SUMOylation appear to be key regulators of VEGFR trafficking. Based on these observations we propose the following hypothesis: VEGF-driven vascular permeability and neovascularization are highly dependent on the targeted subcellular translocation of specific VEGFRs. Pharmacological or genetic manipulation of components of the endosomal trafficking pathway, ?-secretase complex and/or SUMOylation will reduce vascular permeability and inhibit aberrant retinal and choroidal neovascularization. We will test this hypothesis through the following aims. In Aim 1, we will a) determine the origin of nuclear VEGFRs by characterizing the routes of VEGFR internalization and trafficking, b) identify the nuclear targets of VEGFR1 and VEGFR2 and assess how these targets contribute to angiogenesis, c) assess how the ratio of nuclear VEGFR1:VEGFR2 dictates the angiogenic outcome and d) identify the mechanism by which ?- secretase, presenilin and/or sumoylation regulate trafficking of VEGFRs. In Aim 2, we will a) determine if translocation of VEGFRs to AJs and TJs is via membrane diffusion or endosomal trafficking and b) assess how the ratio of VEGFR1 and VEGFR2 at AJs and TJs changes in response to pro- and antiangiogenic factors, the junctional binding partners (e.g. VE-cadherin, ?- catenin, claudin-5) involved and how this affects permeability. In Aim 3, we will a) assess the changes in VEGFR subcellular localization and VEGFR1:VEGFR2 ratio in mice which have undergone pharmacological or genetic modulation of transmembrane proteases and/or SUMOylation and determine if this can prevent VEGF-induced retinal vascular permeability and/or retinal or choroidal angiogenesis in mice and b) evaluate the modulation of novel nuclear signaling pathways identified in sub Aim 1B in mouse models of angiogenesis. Understanding this novel non-canonical VEGF signalling pathway(s) will provide new information on angiogenesis and allow development of a sustainable treatment strategy for AMD and diabetic retinopathy. PUBLIC HEALTH RELEVANCE: Neovascular diseases of the eye include retinopathy of prematurity, proliferative diabetic retinopathy, and the exudative or wet form of age-related macular degeneration (AMD). Together these diseases affect all age groups and are the leading causes of vision impairment in developed nations. The collective evidence suggests that the vascular endothelial growth factor (VEGF) family is critical for ocular angiogensis and this has become a major target for therapeutic intervention. We have identified a novel non-canonical VEGF signalling pathway that requires targeted subcellular translocation of VEGF receptors 1 and 2. We believe that the ratio of VEGFR1:VEGFR2 within subcellular compartments dictates angiogenic outcome and that this is dependent on endosomal sorting and is fine tuned by ?-secretase and sumoylation. Pharmacological or genetic manipulation at different points along this non-canonical pathway will reduce vascular permeability and inhibit aberrant retinal and choroidal neovascularization. Understanding the VEGF signalling pathways will provide new information on the pathogenesis of AMD and diabetic retinopathy and may help develop a sustainable treatment strategy for these debilitating conditions which is a priority area for the NEI.